The present invention relates to methods of delivering and adhering cells to tissue.
In treating damaged skin, such as skin damaged by thermal or chronic wounds, or other wounds, one approach has been to culture keratinocytes derived from biopsies, typically autologous biopsies, until a multilayered, skin-like sheet can be lifted from the culture plate by protease digestion. The difficulties with this process include that the time required to grow such super-confluent cultures, or xe2x80x9cepithelial sheet graftsxe2x80x9d is of the order of three weeks, the thin sheets are difficult to handle, and the xe2x80x9ctake,xe2x80x9d i.e., stable adherence, of the sheet grafts to the basal tissue of the injury has proved problematic. See, e.g., Rennekampff et al., J. Surg Res. 62:288-295, 1996. Moreover, keratinocytes grown to such high density take on the character of non-proliferating, differentiated cells, rather than the actively growing cells that contribute to wound healing. See, e.g., Rennekampff et al. A number of approaches have been undertaken to introduce cultured keratinocytes, to wounds while such cells are in a more active growth phase, as reviewed in Rennekampff et al. These have included growing the cells to subconfluence on porous polymeric supports, and applying the supports, inverted to orientate the cells downwards, onto the wounds. Moreover, certain workers in this field have experimented with mixing keratinocytes with fibrinogen, then mixing in thrombin just prior to application to the wound, so that the fibrinogen is converted to fibrin, which forms a polymeric matrix for the keratinocytes. See, Hundyadi et al., J. Dermatol. Surg. Oncol. 14:75-78, 1988;
Kaiser et al., Burns 20:23-29, 1994. The enzymatic conversion of fibrinogen provides ample time during which the composition has a workable tack, allowing the cell-containing composition to be spread over the wound. The fibrin xe2x80x9cgluexe2x80x9d can also be used to secure a protective layer of allogeneic cadaver skin.
Previous work by the Applicants, together with others working with Applicants, has identified an effective sealant that delivers fibrin, in a xe2x80x9cfibrin monomerxe2x80x9d form that is stabilized against polymerization, to the site that is to be sealed. At the site, the stabilization conditions are reversed, and an effective clot forms. See, Edwardson et al., European Patent Application No. EP 592,242. One ot the particular advantages of tlhis fibrin monomler sealant of EP5 592,242 is that the sealant can be rapidly prepared from a small amount of a patient""s blood only minutes before surgery (or, using manual preparation, within an hour) and this can be done using standard laboratory equipment. Specialized tools for preparing fibrin moniomoier have also been described, and these tools allow an autologous sealant to be prepared from a patient in a rapid, highly reproducible, highly reliable, and highly sate mannier. See, Holm, xe2x80x9cCentriftige Reagent Delivery Systemxe2x80x9d, WO 96/16713, Holm et al., xe2x80x9cMethod and Device for Separating Fibrin I from Blood Plasmaxe2x80x9d, WO 96/16714 and Holm, xe2x80x9cCentrifuge with Annular Filterxe2x80x9d, WO 96/16715. The solubilized fibrin monomer composition can be used as a sealant as described in Edwardson et al., EP 592,242. These improvements thus allow for an autologous sealant to be prepared in a rapid, automated process, and the autologous sealant so prepared is free of extrinsic proteinase enzymes such as bovine thrombin or bovine proteins such as aprotinin.
Another fibrin sealant that can be induced to polymerize without the use of a proteolytic enzyme uses (1) a first component of a fibrin analog where the C-terminal region of the xcex3-chain is sufficiently altered to disrupt self-polymerization and (2) a second component of a fibrin-related molecule such a fibrinogen. See, Cederholm-Williams, WO 9529686A1. On combination of the two components, the fibrin analog polymerizes with the fibrin-related molecule.
Applicants have now shown that keratinocytes sprayed onto a wound site at about the same time frame as the spraying and mixing of two-component fibrin polymer forming systems onto the wound is effective to secure keratinocytes (and, in some cases, fibroblasts) to the wound within a three dimensional fibrin polymer matrix, where the amount of secured cells is effective to expand to form an epithelial layer. In preferred aspects of their invention, the keratinocytes are sprayed concurrently with the spraying and in-flight mixing of a two-component system that renders the fibrin-related molecules of the mixture dynamically competent to polymerize.
The invention also applies to other types of cells that can be secured to a tissue surface, for example to generate new tissue growth or establish the presence of the cells for a sufficient amount of time to achieve a desired result. The invention further relates to spraying cells onto a tissue substrate without fibrin, or with other biocompatible, preferably biodegradable, adhesive polymers. The invention further relates to cells delivered with collagen, with or without flbrin.
The invention provides, among other things, a method of adhering cells to a target surface which is a tissue surface ot a mammal or such a tissue surface coated with a biodegradable polymer sheet, comprising: coating the target surface with a mixture of a first component comprising a non-polymeric fibrin-related protein and a second component effective for converting the fibrin-related protein to fibrin polymer; and spraying a suspension of the cells onto the coated target surface, wherein the mixed two components have formed a fibrin polymer with a tack effective to adhere the cells. Preferably, the fibrin polymer forms in an amount effective to secure a colonization promoting effective amount of the cells on the target surface. In one embodiment, the method further comprises mixing a cell-adherence promoting effective amount of collagen into the mixture. Preferably, the mixture is sprayed to coat the target surface. The mixture and the suspension of cells can be sprayed concurrently to coat the target surface. Preferably, a colonization promoting effective amount of the cells is entrapped in a three-dimensional matrix of fibrin polymer at the target surface.
The method can comprise adhering the biodegradable polymer sheet to the tissue surface, such that the cell suspension is sprayed onto the polymer sheet, which defines the target surface. In one embodiment, the polymer sheet is adhered together with a removable, external backing layer adapted to further restrict, without eliminating, vapor transport from the tissue, and the method further comprises: removing the backing layer after the polymer sheet has adhered to the tissue and thereafter applying the cell suspension. The polymer sheet can, without limitation, comprise a glucosaminglycan polymer sheet or a cross-linked collagen polymer sheet.
The method can further comprise: culturing autologous cells from a biopsy, taken from the mammal, of a tissue of a given type; and forming the cell suspension from the cultured cells, wherein the tissue to which the cells are applied is of the given type or adjacent to tissue of the given type.
In one preferred aspect of the invention, the tissue is a wound and the cell suspension comprises keratinocytes. Preferably, the cultured keratinocytes of the passage producing the cells for the cell suspension are harvested prior to reaching confluence. The method can further comprise: spraying a suspension of the fibroblasts onto the wound coated with the mixed two components. The tibroblasts can be applied to the same mix creating a fibrin polymer, to a separate mix creating the polymer.
In one embodiment, the first component comprises acid-solubilized fibrin, and the second component can comprise an amount of base effective to sufficiently neutralize the mixture to allow the fibrin to polymerize. The application of the first and second components (of any type) can comprise spraying the first and second components such that a stream of the first component merges with a stream of the second component in flight from a spraying device to the surface. The method can comprise spraying the cell suspension, first component and second component such that streams of the cell suspension, first component and second component merge in flight (from a spraying device to the surface).
In one embodiment, the invention provides a method of, in a mammal, delivering cells to a tissue surface, comprising: spraying a suspension of the cells onto the surface in an amount effective to secure a colonization promoting effective amount of the cells on the surface; and maintaining or growing the cells on the surface. Preferably, the cells are sprayed with liquid flows less than 3.0 ml/min. Preferably, the cells are sprayed onto a tissue surface coated with cell-adherence promoting effective amount of collagen, or the cells are sprayed in an gas stream.